This investigation is to examine the toxicity of 5-fluorouracil (FU) and leucovorin (LV) to intestinal epithelial cells. The antitumor activity of FU is due to the incorporation of its anabolite 5- fluorouridine triphosphate (FUTP) into fradulent RNA, and to its other anabolite 5-fluorodeoxyuridylate (FdUMP) which inhibits the synthesis of thymidylate and DNA by forming a ternary complex with the enzyme thymidylate synthase (TS) and the cofactor N-5, 10-methylenetetrahydrofolate (CH2-THF). In some tumor cells, folate analogues by increasing the intracellular CH2-THF concentration enhanced the TS inhibition and cytotoxicity of FU. Phase I-II trials of FU and LV showed that LV significantly improved the response rate of FU in gastric and colorectal cancers, but also potentiated its intestinal toxicity. Anticancer drugs cause intestinal toxicity by inhibiting the mitosis of the proliferating intestinal epithelial crypt cells. The crypt cell kill and recovery is determined by the concentration and duration of tissue exposure to the drug (AUC). The ongoing phase III trials use 10 vastly different regimens of FU and LV, which produce in the intestinal cells different drug AUC's and therefore likely different toxicity to patients. The objectives of this study are to examine the cellular metabolism, pharmacodynamics and mechanism of FU toxicity to the intestinal crypt cells, and the interaction between FU and folates. Preliminary studies, using our newly established cultured intestinal crypt (RIEC) cells derived from normal adult rats, showed that FU was incorporated into RNA, inhibited DNA synthesis and cell growth. The following studies will be done using the RIEC cells. 1. Define the relationship between the cytotoxicity and the AUC of FU alone and FU-folates, using the therapeutic/toxic AUC's from phase III FU-LV protocols. 2. Establish the mechanism of FU toxicity by examining its intracellular metabolism, DNA (inhibition of DNA synthesis) and RNA (incorporation into RNA) effcts, and the modulation of its activity by thymidine and uridine. 3. Compare the kinetics of TS inhibition with those of FdUMP and dUMP. 4. Assess the effects of folates. The proposed studies are expected to illustrate the pharmacology of FU and folates in the intestinal crypt cells, and the reversibility of drug toxicity by thymidine and/or uridine. The comparative cytotoxicity of different AUC's of FU-folates may indicate the relative intestinal toxicity of the different phase III FU-LV regimens.